The current global prevalence of diabetes mellitus is approximately 170 million affected individuals and recent projections suggest this will increase to 300 million worldwide by 2025. More than 30 million individuals are affected in Europe. The majority (around 85%) of patients have type 2 diabetes mellitus (T2D), while some 10-15% of the patients suffer from type 1 diabetes mellitus (T1D). Diabetes causes severe long-term complications and psychosocial problems, imposing a heavy burden of morbidity and premature mortality. Diabetes is the leading cause of blindness and visual disability, limb amputation, end-stage renal failure and neuropathy. It is associated with a greatly increased incidence of cardiovascular disease, including stroke, myocardial infarction and heart failure. Cardiovascular disease accounts for more than 50% of all deaths among diabetic patients in Europe. Because T2D is increasingly prevalent and developing earlier in life, increasing the duration of the disease by decades, many more people are developing severe diabetic complications, and suffering decreases their life quality and expectancy.
Diabetes incurs many costs, and is particularly expensive because it is life-long and causes major medical problems. These costs include direct costs from physician and nursing services, hospital services, laboratory services, drugs, education and training of the patients and indirect costs from time lost from work, chronic nursing and general socio-economic support, early retirement, protracted morbidity and premature mortality.
Overall costs of diabetes mellitus, comprising both direct and indirect costs, have been calculated in various countries and are enormous. For instance, the cost of treating one patient over a 25-year period is in the range of 100,000-200,000 EURO. The present invention, by improving early detection and aiding in the development of novel therapies to prevent and/or revert diabetes, will fulfill an enormous unmet need, opening excellent opportunities for the health care industry and for companies involved in medical imaging, production and distribution of tracers for imaging.
The pancreas, an organ about the size of a hand, is located behind the lower part of the stomach. It comprises two structures that are both morphologically and physiologically different: the exocrine pancreas, which produces the enzymes involved in digestion (amylase, lipase, etc.) and sodium bicarbonate and the endocrine pancreas, which produces the hormones involved in the control of blood glucose (insulin, glucagon, somatostatin and pancreatic polypeptide). The cells of the endocrine pancreas are organized as micro-organs dispersed in the pancreas in the form of islets (islets of Langerhans or pancreatic islets). Each pancreatic islet is made up of 4 cell types: alpha cells, beta cells, delta cells and PP cells. The alpha cells are located at the periphery of the islet and secrete glucagon. The beta cells are found at the center of the islet and are the only cells capable of secreting insulin in response to glucose. The delta cells are at the periphery and secrete somatostatin. The function of the PP cells is more controversial (synthesis of pancreatic polypeptide).
Insulin is a hormone that helps the body use glucose for energy. Diabetes develops when the body doesn't make enough insulin, cannot use insulin properly, or both, causing glucose to build up in the blood. In type 1 diabetes mellitus (T1D)—an autoimmune disease—the beta cells of the pancreas no longer make insulin because the body's immune system has attacked and destroyed them. A person who has T1D must take insulin daily to live. Type 2 diabetes mellitus (T2D) usually begins with a condition called insulin resistance, in which the body has difficulty using insulin effectively. Over time, insulin production declines as well, so many people with T2D eventually need to take insulin.
In addition, a condition called hyperinsulinemia occurs in patients with increased beta cell populations when compared to healthy subjects. Patients with hyperinsulinemia are at high risk of developing seizures, mental retardation, and permanent brain damage. Since glucose is the primary substrate used by the CNS, unrecognized or poorly controlled hypoglycemia may lead to persistent severe neurologic damage. Transient hyperinsulinemia is relatively common in neonates. An infant of a diabetic mother, an infant who is small or large for gestational age, or any infant who has experienced severe stress may have high insulin concentrations. In contrast, congenital hyperinsulinemia is rare.
Among the treatments for diabetes, besides the regular administration of insulin, one of the approaches for the physiological control of glycemia and for normalization of glycemia in diabetics is to restore insulin secretion in vivo from cells. Several strategies have been proposed: xenotransplantation of insulin-producing cells from animals, in vitro differentiation of isolated stem cells into insulin-secreting cells and re-implantation thereof in the patient or allotransplantation of isolated pancreatic islets from another subject.
The lack of a cellular model for studying the beta cells, and also the lack of reliable and effective means of cell sorting suitable for this type of cells hinder the study of beta cell functioning and therefore the development of novel methods of treatment of type I and II diabetes.
The current attempts of imaging pancreatic beta cell mass are being done either by using MRI (magnetic resonance imaging) or by using PET (positron emission tomography) and SPECT (single photon emission computed tomography). In vivo imaging of beta cell mass needs a combination of very high sensitivity and high spatial resolution. MRI has the best spatial resolution but its major problem is the ex-vivo labeling procedure and its semi-quantitative nature. MRI has been successfully used by labeling of human islets with SPIO (small particles of iron oxide) ex vivo and subsequent islet transplantation (Evgenov et al 2006). Using this approach, it was possible to follow up the grafted beta cell mass up to 6 months after transplantation. This technique is, however, only usable for transplantation since it relies on ex vivo uptake of the marker by islet cells and can not be used for in vivo imaging of beta cells in the pancreas. MRI was also used for tracking recruitment of diabetogenic CD8+ T-cells into the pancreas (Moore A et al, 2004), for detecting apoptosis in T1D progression using a Cy5.5 labeled annexin 5 probe (Medarova Z. et al, 2006) or for detection of micro vascular changes in T1D progression (Medarova Z. et al 2007), but the changes detected are semi-quantitative.
PET and SPECT have very high sensitivity and do not require ex-vivo labeling. On the other hand, these techniques have a lower spatial resolution as compared to MRI. PET or SPECT imaging is achieved using islet-specific receptor binding compounds or using compounds taken up specifically by transporters in the pancreatic islets labeled with radioactive tracers. Almost all current substrates used for beta cell PET/SPECT imaging bind or are taken up by the non beta cells and, in some cases, even by exocrine cells in the pancreas. This results in dilution of tracer and high backgrounds, making it currently impossible to quantify the beta cells which are scattered over the pancreas in tiny islets (100-300 μm diameter) constituting only 1-2% of the total pancreas mass. This indicates the urgent need of identification of beta cell specific plasma membrane proteins which can be used for imaging or targeting.
The team of Paul Harris identified 35 islet tissue-restricted transmembrane and membrane-associated molecules by comparing microarray datasets obtained in human islets versus exocrine cells (Maffei et al 2004). One candidate, vesicular monoamine transporter 2 (VMAT2), was selected for additional studies on imaging pancreatic beta cell mass using the specific ligand DTBZ. It was recently shown, however, that total eradication of beta cells still resulted in VMAT2 binding, showing that DTBZ is not a good biomarker for imaging pancreatic beta cell mass (Kung et al 2007).
One of few beta-cell specific membrane proteins identified up to now is the zinc transporter ZnT8 or SLC30A8 (Chimienti F et al 2004, Seve et al 2004). ZnT8 co-localizes with insulin in the pancreatic beta cells (Chimienti et al 2006). Avalon (EP1513951 and WO03097802) and CEA (patent US2006246442, EP1563071 and WO2004046355) introduced patents on the fact that this protein was beta cell specific and on the usage of an antibody against SLC30A8 or ZnT8 for cancer therapy and for use in antibody test.
Recently SLC30A8 was identified as an autoantigen and the target of autoantibodies in type 1 diabetes (Wenzlau J M et al., 2007) and it is therefore not useful for beta cell detection.
The company Biogen-IDEC identified Kirrel 2 (filtrin or NEPH3) an immunoglobulin superfamily gene which is specifically expressed in the beta cells of pancreatic islet cells (Sun C. et al, 2003) and in kidney (Rinta-Valkama J et al 2007). Due to its very low expression levels this candidate is not useful for beta cell detection. Recently it was shown that densin and filtrin can act as auto-antigens and auto-antibodies against these are detected in T1D patients (Rinta-Valkama J et al 2007). However, this candidate was too low expressed to be of use in beta cell detection.
Tmem27 or collectrin was identified as a beta cell protein that stimulates beta cell proliferation (Fukui K et al, 2005) and which is cleaved and shed from the plasma membrane. It was not retained in our biomarker list since its expression is higher in the islet non beta cells than in the beta cells.
The free fatty acid receptor GPR40 (also called FFAR1) is a G-coupled receptor recently identified as islet specific and as a possible target for treatment of T2D (Bartoov-Shifman R et al 2007). It was recently suggested to be a possible candidate biomarker for imaging pancreatic beta cells. This receptor, however, is expressed both in islet beta cells and in alpha cells (Flodgren E et al 2007), hampering its potential as a good beta cell biomarker.
The fatty acid receptor GPR119 was identified as a beta-cell specific receptor (Frederiksson R et al, 2003, Chu et al 2007) and oleoylethanolamide (OEA)/lysophosphatidylcholine (LPC)-activated GPR119 is involved in glucose induced insulin secretion. Whether or not these metabolite receptors reach sufficiently high concentrations on the outer surface of the plasma membrane to image the beta cell mass needs to be determined (Madiraju S R et al 2007). GPR119 was identified recently in islet non beta cells (Sakamoto Y et al, 2006) and the selective small-molecule GPR119 agonist PSN632408 suppressed food intake, reduced body weight gain and white adipose tissue deposition (Overton H A et al, 2006) showing that GPR119 is expressed in other tissues.
A random phage-displayed 20-mer peptide library was screened on freshly isolated rat islets but none of the selected peptides were selectively enough in binding to the islets versus other tissues to be used for imaging (Samli K N et al 2005).
PET imaging teams working on pancreas are also attempting to image beta cells. For this purpose, they are using compounds assumed to selectively bind or being taken up by islet-specific transporters and receptors. Examples of these compounds include glibenclamide, tolbutamide, serotonin, L-DOPA, dopamine, nicotinamide, fluorodeoxyglucose, and fluorodithizone. Glibenclamide and fluorodithizone are not specific enough to attain the robust signal to background ratio needed for quantification of beta cell mass via PET imaging. F-deoxy glucose (FDG) could not be used to successfully quantify beta cell mass (Malaisse W J et al. 2000, Ruf J et al. 2006, Nakajo M. et al., 2007) but could be used to discriminate between focal and diffuse hyperinsulinism (de Lonlay P et al 2005 and 2006, Otonkoski T et al 2006, Kauhanen S et al 2007, Ribeiro M J et al, 2007, Hardy O T., et al 2007).
The most promising compounds used up to now to image pancreatic beta cells are F18-DOPA and Dihydrotetrabenazine (DTBZ), both substrates taken up by the VMAT2 transporter (Souza F. et al 2006, Simpson N R. et al. 2006) and Glucagon-like peptide 1 (GLP-1) or exendin (ligands binding to the GLP-1 receptor (Gotthardt M. et al, 2002, Wild M. et al. 2006). Unfortunately, all the compounds mentioned above result in too high background levels and non-specific binding to various other intra-abdominal tissues such as kidney and liver.
As described above, the team of Paul Harris identified vesicular monoamine transporter 2 (VMAT2) and its ligand DTBZ as potential tools for beta cell imaging. DTBZ was labeled with C-11 and F-18 and a high pancreatic uptake was obtained in rodents and primates (Souza et al, 2006). Unfortunately, complete eradication of beta cells reduced the pancreatic uptake of DTBZ by only 30-40% showing that the compound lacks sufficient specificity for the beta cells (Kung et al 2007) to enable its use to assess beta cell mass.
Several auto-antibodies directed against insulin (K14D10), sulfatide (102), glutamic acid decarboxylase (GAD) or protein tyrosine phosphatase (IA2) have been identified. The team of Ian Sweet used a beta cell specific antibody (K14D10) and its Fab fragment for imaging/targeting beta cells but the antibody fragment with the best blood clearance failed to preferentially accumulate in the pancreas. The monoclonal antibody IC2 (Brogren C H et al 1986, Buschard K et al 1988), modified with a radioisotope chelator for nuclear imaging, showed highly specific binding and accumulation to beta-cells with virtually no binding to exocrine pancreas or stromal tissues (Moore A et al, 2001). Sulfatide, however, is also expressed in islet cell innervating Schwann cells and other neural tissues, which may hamper its use in beta cell imaging.
Consequently, there is a lack of specific and reliable markers for the beta cells of pancreatic islets of Langerhans and do not allow reliable beta cell mass quantification. One of the aims of the present invention is to provide such markers.
Our candidates are specific for beta cells and can be used for beta cell specific targeting and non-invasive imaging. They are not induced by inflammation and are not expressed in pancreas surrounding tissues. The use of targeting strategies against these biomarkers will allow early identification of loss in beta cell mass and the follow up of therapies for diabetes, including islet transplantation, attempts at beta cell regeneration etc.